Association of Diaporthe longicolla with Black Zone Lines on Mature Soybean Plants

In 2014, during a survey for soybean (Glycine max L.) diseases in Illinois, Indiana, Iowa, Michigan, and South Dakota, zone lines were observed on the lower stems of soybean plants. The survey was performed by sampling two to three fields per state. In each field, at least five plants exhibiting zone lines were collected. Isolations were made from the zone lines by plating 1-cm pieces on potato dextrose agar. A total of 90 isolates producing black stromata in concentric patterns and alpha conidia were tentatively identified as Diaporthe longicolla (Hobbs) Santos, Vrandecic and Phillips. DNA was extracted from the mycelia of 10 representative isolates and the identity was confirmed by sequencing the internal transcribed spacer (ITS) region. Additionally, phylogenetic analysis combining translation elongation factor-1a and actin sequences was performed and the ten isolates grouped with the D. longicolla ex-type cultures in a well-supported clade (94% bootstrap support). A pathogenicity test was performed in the greenhouse by inserting D. longicolla-infested toothpicks into the lower stems of the soybean plants. To complete Koch's postulates, D. longicolla was re-isolated from the zone lines of the inoculated plants and the pathogen identity was confirmed by sequencing the ITS gene.